Polycyclic ether antibiotic

ABSTRACT

A new acidic polycyclic ether antibiotic UK-58,852 has the formula: ##STR1## wherein R and R 1  are both hydrogen, and can be prepared by the submerged aerobic propagation in aqueous nutrient media of Actinomadura sp. ATCC 39697. The antibiotic and its cationic salts are active against a variety of microorganisms and are effective in controlling coccidiosis, enteritis, swine dysentery and theileriosis as well as being effective in promotion of growth and/or improving efficiency of feed utilization in swine and ruminants. Two minor components, wherein R is H and R 1  is CH 3  and wherein R and R 1  are both CH 3 , have also been isolated from the fermentation.

BACKGROUND OF THE INVENTION

This invention is concerned with new members of the acidic polycyclicether group of antibiotics, a class of compounds characterizedbiologically by their effect on cation transport in mitochondria. Thisfamily of antibiotics includes such well known agents as monensin;nigericin; grisorixin; dianemycin; salinomycin; mutalomycin; ionomycinand leuseramycin. The subject has been reviewed by Westley, "PolyetherAntibiotics", Adv. Appl. Microbiol., 22, 177, 1977.

The polycyclic ether antibiotics listed above are active againstGram-positive bacteria, fungi and protozoa. In particular theseantibiotics exhibit potent anti-coccidial activity. They have thereforebeen employed with varying degrees of success in the treatment of avariety of animal infections.

The well-known protozoan disease, coccidiosis, continues to be a seriousproblem and its control is of economic importance to veterinary science,especially to the poultry industry. Coccidiosis results from infectionby one or more species of Eimeria or Isospora (for a summary, see Lundand Farr in "Diseases of Poultry," 5th ed., Biester and Schwarte, Eds.,Iowa State University Press, Ames, Ia., 1965, pp. 1056-1096). There aresix species of coccidia which produce easily discernible morbidity insusceptible chickens. Eimeria tenella, E. necatrix, E. brunetti, E.acervulina, E. maxima and E. mivati produce damage either directlythrough destruction of epithelial cells of the digestive tract orindirectly through production of toxins. Three other species of protozoabelonging to the same genus are considered to be relatively innocuous;however, E. mitis, E. hagani and E. praecox are capable of reducingweight gain, lowering feed efficiency and adversely affecting eggproduction.

In view of the great economic losses due to coccidiosis and the knowndisadvantages of existing anti-coccidial agents, the search for betteranticoccidial agents continues.

Enteritis is another disaease which can cause severe economic losses tolivestock producers. Enteritis occurs in chickens, swine, cattle andsheep and is attributed mainly to anaerobic bacteria, particularlyClostridium perfringens, and viruses. Enterotoxemia in ruminants, anexample of which is "overeating disease" in sheep, is a condition causedby C. perfringens infection.

Swine dysentery is one of the most common swine diseases diagnosed inthe United States. Additionally, the disease is prevalent in many othercountries and annually causes considerable losses in stock to swinegrowers around the world. It has recently been discovered that a largespirochete is the causative organism of the disease. This organism,Treponema hyodysenteriae, has now been isolated and shown to be capableof producing the disease [Harris, D. L. et al. "Swine-Dysentery-1,Inoculation of Pigs with Treponema hyodysenteriae (New species) andReproduction of the Disease," Vet. Med/SAC, 67, 61-64, 1972]. The testdata recited hereinafter concerns tests conducted with this organism. Itmust be noted that it is not known whether T. hyodysenteriae is the solecausative organism of swine dysentery. From the data available, however,it can be concluded that it is a primary source of the infection.

Performance enhancement (increased rate of growth and/or increasedefficiency of feed utilization) in ruminants such as cattle, and inmonogastric animals such as swine, is another economically desirableobjective of veterinary science. Of particular interest is improvedperformance achieved by increasing the efficiency of feed-utilization.The mechanism for utilization of the major nutritive portion of ruminantfeeds is well known. Micro-organisms in the rumen of the animal degradecarbohydrates to produce monosaccharides and then convert thesemonosaccharides to pyruvate compounds. Pyruvates are metabolized bymicrobiological processes to form acetates, butyrates or propionates,collectively known as volatile fatty acids. For a more detaileddiscussion, see Leng in "Physiology of Digestion and Metabolism in theRuminant," Phillipson et al., Eds., Oriel Press, Newcastle-upon-Tyne,England, 1970, pp. 408-410.

The relative efficiency of volatile fatty acid utilization is discussedby McCullough in "Feedstuffs", June 19, 1971, page 19; Eskeland et al.in J. An. Sci., 33, 282, 1971; and Church et al. in "DigestivePhysiology and Nutrition of Ruminants", Vol. 2, 1971, pp. 622 and 625.Although acetates and butyrates are utilized, propionates are utilizedwith greater efficiency. Furthermore, when too little propionate isavailable, animals may develop ketosis. A beneficial compound,therefore, stimulates animals to produce a higher proportion ofpropionates from carbohydrates, thereby increasing carbohydrateutilisation efficiency and also reducing the incidence of ketosis.

Yet another disease which causes economic losses to live-stock producersis caused by the protozoan parasite Theileria parva. That disease,theileriosis, is also known as "East Coast fever", "Coastal fever" or"Rhodesian tick fever". The Theileria parasite invades but does notdestroy red blood cells which gives rise to acute or chronic febrileinfections. In cattle the disease is characterized by high fever,swelling of the lymph nodes, emaciation and high mortality. The diseaseis a very serious problem in East and Central Africa. See further "TheMerck Veterinary Manual", Siegmund et al., Eds., Merck & Co., Rahway,N.J., 5th Ed., pp. 431-433 (1979).

SUMMARY OF THE INVENTION

This invention is concerned with a new acidic polycyclic etherantibiotic designated UK-No. 58,852, produced by the submerged aerobicpropagation in aqueous nutrient media of the microorganism Actinomaduraroseorufa Huang sp. nov., ATCC 39697 isolated from a soil sample fromJapan. The antibiotic and its cationic salts are active against avariety of microorganisms and are effective in controlling coccidiosis,enteritis, swine dysentery and theileriosis as well as being effectivein promoting growth and increasing efficiency of feed utilisation inswine and ruminants. The invention also includes two related minorcomponents produced in the fermentation (designated CP-70,228 andCP-70,828) which are also antibiotics effective in controllingcoccidiosis. CP-70,228 is also useful in promoting growth and increasingefficiency of feed utilization in swine and ruminants.

DETAILED DESCRIPTION OF THE INVENTION

The microorganism designated Actinomadura roseorufa Huang sp., nov.,ATCC 39697, which is useful for the preparation of antibiotics UK-No.58,852, CP-70,228 and CP-70,828 was isolated from a soil samplecollected in Izumo City, Shimane Prefecture, Japan. It was recognized tobe an Actinomadura species because of the narrow dimensions of thehyphae, production of aerial mycelium, spores being borne in chains andpresence of madurose in the whole-cell hydrolysates.

A culture thereof, designated herein as N596-33, was plants from a slantinto ATCC no. 172 broth and grown for four days at 28° C., on a shaker.It was then centrifuged for 20 minutes, washed three times with steriledistilled water and planted on media commonly used for identification ofmembers of the Actinomycetales as hereinafter described.

The culture was incubated at 28° C. and the results were read at varyingtimes but most commonly were taken at 14 days. The colours are describedin common terminology, but exact colours were determined by comparisonswith colour chips from the Colour Harmony Manual, fourth edition. Themethods of whole-cell amino acid and sugar analyses are those describedin Becker et al., Appl. Microbiol., 12, 421-423, 1964; and inLechevalier, J. Lab. Clin. Med., 71, 934-944, 1968.

Identification media used for the characterization of the culture andreference for their composition or supplier are as follows:

1. Yeast Extract-Malt Extract Agar--(ISP medium no. 2, Difco).

2. Oatmeal Agar--(ISP medium no. 3, Difco).

3. Inorganic Salts-Starch Agar--(ISP medium no. 4, Difco).

4. Glycerol-Asparagine Agar--(ISP medium no. 5, Difco).

5. Czapek-Sucrose Agar--S. A. Waksman, The Actinomycetes, Vol. 2, mediumno. 1, p. 328, 1961.

6. Glucose-Asparagine Agar--Ibid, medium no. 2, p. 328.

7. Bennett's Agar--Ibid, medium no. 30, p. 331.

8. Emerson's Agar--Ibid, medium no. 28, p. 331.

9. Nutrient Agar--Ibid, medium no. 14, p. 330.

10. Gordon and Smith's Tyrosine Agar--R. E. Gordon and M. M. Smith, J.Bact., 69, 147-150, 1955.

11. Casein Agar--Ibid.

12. Calcium Malate Agar--S. A. Waksman, Bact. Rev. 21, 1-29, 1957.

13. Gelatin Agar--R. E. Gordon and J. M. Mihm, J. Bact., 73, 15-27,1957.

14. Starch Agar--Ibid.

15. Potato Carrot Agar--M. P. Lechevalier, J. Lab. and Clinical Med.,71, 934-944, 1968, but use only 30 g potatoes, 2.5 g carrots and 20 gagar.

16. 2% Tap Water Agar.

17. Gauze's Mineral Medium no. 1--G. F. Gauze et al., Problems in theClassification of Antagonistic Actinomycetes. English Ed., p. 13, 1957.

18. Gauze's Organic Medium no. 2--Ibid.

19. Potato Dextrose Agar--Peel, cut up and steam 100 g. potatoes in 500ml water, filter through cheese cloth, add 10 g. glucose, 50 ml coconutmilk, 20 g. agar and enough water to make one liter.

20. Tryptone-Yeast Extract Broth--(ISP medium no. 1, Difco).

21. Peptone-Yeast Extract Iron Agar--(ISP medium no. 6, Difco).

22. Organic Nitrate Broth--R. E. Gordon and J. M. Mihm, J. Bact., 73,15-27, 1957.

23. Dextrose Nitrate Broth--S. A. Waksman, The Actinoymcetes, Vol. 2,medium no. 1, p. 328, 1961, with 3 g dextrose substituted for 30 gsucrose and agar omitted.

24. Skim Milk--Difco.

25. Cellulose utilization

(a) H. L. Jensen, Proc. Linn. Soc. N.S.W., 55, 231-248, 1930.

(b) M. Levine and H. W. Schoenlein, A Compilation of Culture Media,medium no. 2511, 1930.

26. Carbohydrates--ISP medium no. 9, Difco; C-2 medium, H. Nonomura andY. Ohara, J. Ferment. Technol., 49 (11), 887-894, 1971.

27. Temperature Range--ATCC medium 172 in ATCC Culture CollectionCatalogue, 15th ed., p. 608, 1982.

The observations of growth and appearance of the organism were asfollows:

Yeast Extract-Malt Extract Agar--Growth good, white, with pink topink-red tint (6ga, 61/2ea, 61/2ia), raised, wrinkled, aerial myceliumwhite; reverse pale yellowish to pink (2ea., 6ga); no soluble pigment.

Oatmeal Agar--Growth moderate, cream (11/2ca), slightly raised, smooth;aerial mycelium none to sparse, white; reverse colourless to cream(11/2ca); no soluble pigment.

Inorganic Salts-Starch Agar--Growth poor, colourless to pale cream (near11/2ca), thin, smooth; aerial mycelium none to sparse, white; reversecolourless to pale cream (near 11/2ca); no soluble pigment.

Glycerol-Asparagine Agar--Growth poor to moderate, colourless but palepink (5ca) near the edge, thin but moderately raised near the edge,smooth; aerial mycelium none to sparse, white; reverse colourless topale pink (5ca); no soluble pigment.

Czapek-Sucrose Agar--Growth moderate, pale pink, grayish yellow to red(5ca, 2gc, 61/2ia), slightly raised, smooth; aerial mycelium none tosparse, visible only under the microscope; reverse same as surface; nosoluble pigment.

Glucose-Asparagine Agar--Growth moderate to good, grayish yellow,yellowish brown to red (2ic, 3nc, 71/2ia, 71/2la), moderatedly raised,smooth, with a few small bumps; aerial mycelium none to sparse, white;reverse same as surface; soluble pigment cream (2ca).

Bennett's Agar--Growth good, grayish yellow (2gc, 2ie) with a pale pinkedge (5ca), raised, wrinkled, with cracks in some areas; aerial myceliumnone to sparse, white; reverse same as surface; soluble pigment grayishyellow (2ic).

Emerson's Agar--Growth good, pale yellowish, gray to dark brown (2gc,near gray series 3fe, 3li, 3ni), raised, wrinkled; aerial myceliumshort, white; reverse dark brown to yellowish (3li, 4li, 2gc); solublepigment yellowish (2lc).

Nutrient Agar--Growth moderate, orange (5ea, 5ga), thin to slightlyraised, smooth; aerial mycelium none to sparse, white; reverse orange(5ga); no soluble pigment.

Gordon and Smith's Tyrosine Agar--Growth moderate to good, brown to darkbrown (3le, 3lg, 3ni), moderately raised, smooth to slightly granular,with a crack in one streak; aerial mycelium none to sparse, white;reverse same as surface; soluble pigment yellowish brown (3lc).

Casein Agar--Growth moderate to good, pale pink (5ca, 5ea), slightlyraised, smooth to wrinkled; aerial mycelium none or sparse near theedge, white; reverse pale yellowish to pale pink (2ca, 2ea, 5ca);soluble pigment yellowish brown (3lc).

Calcium Malate Agar--Growth moderate, cream (2ca) to white, appearing asisolated colonies, raised, smooth, aerial mycelium white; reverse cream(2ea, 2ca); no soluble pigment.

Gelatin Agar--Growth moderate, cream to dark yellowish (2ea, 2ic),slightly raised, smooth to slightly granular; aerial mycelium none tosparse, white; reverse pale yellowish (2ea); no soluble pigment.

Starch Agar--Growth moderate to good, cream (2ca, 2ea), moderatelyraised, smooth to slightly granular; aerial mycelium none to sparse,white; reverse cream (2ca, 2ea); no soluble pigment.

Potato Carrot Agar--Growth moderate, cream (2ca), slightly raised,smooth; aerial mycelium none to sparse, white; reverse cream (2ca); nosoluble pigment.

Tap Water Agar--Growth poor, colourless to pale cream (11/2ca), thin,smooth, no aerial mycelium; reverse colourless; no soluble pigment.

Gauze's Mineral Medium 1--Growth moderate, cream, yellowish green topink-red (2ca, 11/2gc, 11/2ic, 5ca, 6ia, 6ga, 6la), thin, smooth, with afew spots of white aerial mycelium; reverse same as surface; no solublepigment.

Gauze's Organic Medium 2--Growth moderate to good, pink-orange (4ea),moderately raised, smooth but slighlty wrinkled near the edge; aerialmycelium short, white; reverse same as surface; no soluble pigment.

Potato Dextrose Agar--Growth good, dark yellowish, pink to red (2ic,6ea, 61/2nc), raised, wrinkled, aerial mycelium white to pink (6ea);reverse dark yellowish, red to dark red (2ic, 61/2nc, 61/2ni); solublepigment pale yellowish (2ea).

Morphological Properties--The following morphological properties wereobserved on inorganic salts-starch agar after 21 days of incubation:spore mass in white colour-series; sporophores monopodially branched;spore chains flexuous or wavy, occasionally hooked, irregularly curved,or very loosely coiled up to 3 turns, 10 to 30 spores per spore chain;spores oval to elliptical, rarely globose, 1.1-1.6×0.7-1.0 μm or 0.7-1.0μm in diameter; warty, as revealed by scanning electron microscopy.

Biochemical Properties--Melanin not produced in tryptone-yeast extractbroth; hydrogen sulfide not produced on peptone-yeast extract iron agar;gelatin liquefied; starch not hydrolyzed; nitrate reduced to nitrite inboth organic nitrate broth and dextrose nitrate broth; no growth and nodecomposition on both cellulose media; clearing and no coagulation onmilk; casein digestion positive; digestion of calcium malate positive;tyrosine digestion weakly positive. Carbohydrate utilization was thesame on ISP medium No. 9 and Nonomura and Ohara's C-2 medium: Glucoseand rhamnose utilized; sucrose weakly utilized; fructose doubtfullyutilized; arabinose, inositol, mannitol, raffinose and xylose notutilized.

Whole-Cell Analysis--The whole-cell hydrolysates containmeso-diaminopimelic acid, madurose, galactose, glucose, rhamnose andribose.

Temperature Relations--The relationship of temperature to growth ratewas observed to be as follows:

    ______________________________________                                        21° C.                                                                         28° C.                                                                             37° C.                                                                            45° C.                                  ______________________________________                                        Good    Good        Moderate to                                                                              No growth                                      growth  growth      good growth                                               ______________________________________                                    

The micro-organism is characterized by its inability to produce melanin,flexuous to wavy spore chains with warty spores, and presence ofmeso-diaminopimelic acid and madurose as whole-cell components. Theaerial mycelium if produced is rudimentary and on some media is visibleonly under the microscope. The colour of aerial mycelium is white butmay be pink on potato dextrose agar. The vegetative mycelium isdistinctive, showing some shade of red on yeast extract-malt extractagar, Czapek-sucrose agar, glucose-asparagine agar, Gauze's organicmedium no. 1 and potato dextrose agar. The culture fails to producespores on most of the media used. On inorganic salts-starch agar,oatmeal agar and Gauze's mineral medium no. 1 they are produced in theform of a few, small, raised, white patches. The morphological andbiochemical properties place the micro-organism in the genusActinomadura. However the organism is different from any related speciesand represents a new species of the genus. It is herein designated asActinomadura roseorufa Huang sp. nov. The specific epithet refers to thepink, pink-red, or red substrate mycelium of the culture. The culturethereof, N596-33, has been deposited with the American Type CultureCollection; 12301 Parklawn Drive, Rockville, Md 20852, U.S.A. under theprovisions of the Budapest Treaty on 24th May, 1984 under the AccessionNumber ATCC 39697.

Cultivation of Actinomadura roseorufa Huang sp. nov., ATCC 39697 andisolation of the Antibiotics UK-58,852, CP-70,228 and CP-70,828 may beconducted under conditions similar to those employed in previousfermentations yielding polyether antibiotics. See, for example, U.S.Pat. No. 4,361,649. Cultivation preferably takes place in aqueousnutrient media under submerged aerobic conditions with agitation at atemperature of 24° to 36° C. Nutrient media useful for cultivationinclude a source of assimilable carbon such as sugars, starches andglycerol; a source of organic nitrogen such as casein, enzymatic digestof casein, soybean meal, cotton seed meal, peanut meal, wheat gluten,soy flour, meat meal and fish meal. A source of growth substances suchas grain solubles, fishmeal, cotton seed meal, and yeast extract as wellas mineral salts such as sodium chloride and calcium carbonate and traceelements such as iron, magnesium, copper, zinc, cobalt and manganese mayalso be utilized with advantageous results. If excessive foaming isencountered during fermentation, antifoam agents such as vegetable oilsor silicones may be added to the fermentation medium. Aeration of themedium in tanks for submerged growth is preferably maintained at therate of about 1/2 to 2 volumes of sterile free air per volume offermentation broth per minute forced into the broth through a sparger.Agitation may be maintained by means of agitators generally familiar tothose skilled in the fermentation art. The rate of agitation depends onthe type of agitator employed. A shake flask is usually run at 150 to200 cycles per minute whereas a fermentor is usually run at 300 to 1700revolutions per minute. Aseptic conditions must, of course, bemaintained through the transfer of the organism and throughout itsgrowth.

Inoculum for the preparation of the antibiotics according to thisinvention may be obtained by employing growth from a slant of theculture or Roux bottles inoculated with the culture. A solid mediumsuitable for initial growth of the organism on slants and in Rouxbottles is ATCC medium no. 172. The growth may be used to inoculateeither shake flasks or inoculum tanks or the inoculum tanks may beseeded from the shake flasks. Growth in shaken flasks will generallyhave reached its maximum in 4 to 5 days whereas inoculum in submergedinoculum tanks will usually be in the most favourable period in 5 to 6days.

The progress of antibiotic production during fermentation and thebioactivity of the fermentation broth can be monitored by biologicalassay of the broth employing a sensitive strain of Staphylococcus aureusor Bacillus subtilis. B. subtilis ATCC 6633 is a suitable strain forthis purpose. Standard plate assay technique is employed in which thezone of inhibition surrounding a filter paper disc saturated with thebroth is used as a measure of antibiotic potency. Also, thin-layerchromatogrpahy employing silica gel is a useful tool for detecting theantibiotics produced in fermentation media and analyzing the compositionof crude and purified materials extracted from the fermentation broths.The chromatograms are developed with ethyl acetate and the antibioticcompounds are visualized by spraying with vanillin reagent and heatingthe TLC plate at 80° C. The developed plate can also be overlayed withagar seeded with either S. aureus or B. subtilis and incubated at 37° C.for 16 hours to visualize the antibiotics.

The Antibiotic UK-58, 852 produced by fermentation of Actinomaduraroseorufa Huang sp. nov., ATCC 39697 may be separated and recovered byextracting the whole, unfiltered fermentation broth with an organicsolvent such as chloroform, ethyl acetate, methylisobutyl ketone orbutanol at the naturally prevailing pH. Alternatively the mycelium canbe separated after growth has been completed and the mycelium extractedwith an organic solvent. The solvent extract can then be concentrated toa thin syrup and the pure antibiotic is obtained by chromatography.

The minor components CP-70,228 and CP-70,828 are recovered by furtherchromatography of the mother liquors.

A typical method of separation and recovery of the antibiotic compoundof this invention is as follows:

The whole broth from fermentation of Actinomadura roseorufa Huang sp.nov., ATCC 39697 was extracted with methylisobutyl ketone. The solventextract yielded a dark oil on solvent evaporation under vacuum. The oilwas dissolved in hexane and poured onto a bed of silica gel. The silicagel bed was washed repeatedly with hexane and then eluted withchloroform, chloroform/ethyl acetate and ethyl acetate. The eluates wereexamined by thin-layer chromatography. Fractions containing theAntibiotic UK-No. 58,852 were combined and evaporated to dryness. Theproduct fraction may be further purified by crystallisation or by columnchromatography if desired. Cycloserine is co-produced with theAntibiotic UK-No. 58,852 but, because of its poor solvent solubility itdoes not interfere with the recovery of Antibiotic UK-No. 58,852.

The minor components CP-70,228 and CP-70,828 are recovered by furtherelution of the column using a more polar solvent system. The crudeproducts may be purified by further chromatography as necessary. Inaddition, the antibiotic CP-70,228 is produced when the free acid formof UK-No. 58,852 is allowed to stand in methanol solution.

The antibiotic compounds of this invention are acidic, and will formcationic salts by reaction with basic agents. All such salts are withinthe scope of this invention. These salts are prepared by conventionalmethods for polyether (ionophore) antibiotics. In one method, a solutionof the antibiotic in a volatile, water immiscible, organic solvent iswashed with an aqueous solution containing at least a stoichiometricequivalent, and preferably a large excess, of an appropriate basicagent. After drying the organic solvent solution it is evaporated invacuo to give the desired cationic salt. Typical basic agents which canbe used for this purpose include alkali metal hydroxides, such as sodiumhydroxide and potassium hydroxide, alkaline earth metal hydroxides, suchas calcium hydroxide and barium hydroxide, and ammonium hydroxide.

X-ray analysis of the silver salt of Antibiotic UK-No. 58,852 andanalytical and spectral data for CP-70,228 and CP-70,828 indicates thatthe compounds have the following structure: ##STR2## UK-58,852: R=H, R¹=H CP-70,228: R=H, R¹ =CH₃

CP-70,828: R=CH₃, R¹ =CH₃.

Antibiotic UK-58,852 exhibits inhibitory action against the growth of anumber of Gram-positive microorganisms. In Table I, below, the resultsof in vitro tests are summarized. For this test each organism isinoculated in a series of test tubes containing nutrient medium andvarying concentrations of Antibiotic UK-58,852 to determine the minimalconcentration of the compound in mcg./ml. which inhibits the growth ofthe organism over a period of 24 hours (MIC).

                  TABLE I                                                         ______________________________________                                        ANTIBACTERIAL ACTIVITY                                                                       Strain   MIC, mcg./ml. Antibiotic                              Organism       No.      UK-58,852 (sodium salt)                               ______________________________________                                        Staphylococcus aureus                                                                        01A005   3.12                                                                 01A052   3.12                                                                 01A110   3.12                                                                 01A400   6.25                                                  Streptococcus faecalis                                                                       02A006   >50                                                   Streptococcus pyogenes                                                                       020303   0.05                                                  Actinomyces pyogenes                                                                         14D011   <0.08                                                 Actinobacillus 44B004   >25                                                   pleuropneumoniae                                                              Pasteurella multocida                                                                        59A006   >200                                                  Clostridium perfringens                                                                      10A009   0.79                                                  Bacteroides fragilis                                                                         78C024   0.10                                                  Fusobacterium  84C004   >25                                                   necrophorum                                                                   Treponema hyodysenteriae                                                                     94A007   0.39                                                  ______________________________________                                    

Against the gram-negative bacteria such as Escherischia coli,Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens andEnterobacteriaceae aerogenes, MIC values were >50 in each case.

Antibiotic UK-58,852 and its cationic salts exhibit excellent activityagainst coccidial infections in poultry. When incorporated into the dietof chickens at levels of 25 to 150 ppm, these compounds are effective incontrolling infections due to Eimeria tenella, E. acervulina, E. maxima,E. brunetti and E. necatrix.

Efficacy data for Antibiotic Compound UK-58,852 and its salts againstcoccidial infections in chickens was obtained in the following fashion.Groups of 3-5 ten-day old pathogen free white leghorn cockerel chickswere fed a mash diet containing Antibiotic UK-58,852 or its sodiumand/or potassium salt uniformly dispersed therein. After being on thisration for 24 hours each chick was inoculated per os with oocysts of theparticular species of Eimeria being tested. Other groups of 3-5 ten-dayold chicks were fed a similar mash diet without Antibiotic UK-58,852 orits salts. They were also infected after 24 hours and served as infectedcontrols. Yet another group of 3-5 ten-day old chicks were fed the samemash diet without Antibiotic UK-58,852 and were not infected withcoccidia. These served as normal controls. The results of treatment wereevaluated after five days in the case of E. acervulina, and six days forall other challenges.

The criteria used to measure anticoccidial activity consisted of lesionscores of 0 to 4 for E. tenella after J. E. Lynch, "A New Method for thePrimary Evaluation of Anticoccidial Activity", Am. J. Vet. Res., 22,324-326, 1961; and 0 to 3 for the other species based on modification ofthe scoring system devised by J. Johnson and W. H. Reid, "AnticoccidialDrugs. Lesion Scoring Techniques in Battery and Floor Pen Experiments inChicks", Exp. Parasit., 28, 30-36, 1970. A constant ratio wasestablished by dividing the lesion score of each treated group by thelesion score of the infected control.

Table II summarizes the results obtained.

                  TABLE II                                                        ______________________________________                                        IN VIVO ANTICOCCIDIAL ACTIVITY                                                           DOSE     AVERAGE                                                   INFECTION  (PPM IN  DEGREE OF   WEIGHT GAIN                                   SPECIES    FEED)    INFECTION   (PERCENT)                                     ______________________________________                                        Eimeria tenella                                                                          150      0.0          0                                                       125      0.0          4                                                       100      0.0          8                                                        75      0.0         28                                                        50      0.0         45                                                        25      0.0         93                                            Eimeria acervulina                                                                       150      0.0          0                                                       125      0.0          0                                                       100      0.0         23                                                        75      0.0          0                                                        50      0.0         62                                                        25      0.0         98                                            ______________________________________                                    

The value of animal feeds has generally been determined directly byfeeding the animal. British Patent Specification No. 1,197,826 detailsan in vitro rumen technique whereby the changes occurring in feedsbrought about by microorganisms are measured more readily and with greataccuracy in the evaluation of animal feeds. This technique involves theuse of an apparatus in which the digestive processes of the animals areconducted and studied in vitro. The animal feeds, rumen inoculum andvarious growth promotants are introduced into and withdrawn from alaboratory unit under carefully controlled conditions and the changestaken place are studied critically and progressively during theconsumption of the feed by the microorganisms. An increase in thepropionic acid content of the rumen fluid indicates that a desirableresponse in overall ruminant performance has been brought about by thegrowth promotant in the feed composition. The change in propionic acidcontent is expressed as percent of the propionic acid content found inthe control rumen fluid. Long term in vivo feeding studies are used toshow a reliable correlation between propionic acid increase in the rumenfluid and improved animal performance.

Rumen fluid is collected from a fistulated cow which is fed on acommercial fattening ration plus hay. The rumen fluid is immediatelyfiltered through cheese cloth, and 10 ml. added to a 50 ml. conicalflask containing 400 mg. of standard substrate (68% corn starch+17%cellulose+15% extracted soybean meal), 10 ml. of a pH 6.8 buffer and thetest compound. The flasks are gassed with oxygen free nitrogen for abouttwo minutes, and incubated in a shaking water bath at 39° C. for about16 hours. All tests are conducted in triplicate.

After incubation, 5 ml. of the sample is mixed with 1 ml. of 25%metaphosphoric acid. After 10 minutes 0.25 ml. of formic acid is addedand the mixture centrifuged at 1500 rpm for 10 minutes. Samples are thenanalyzed by gas-liquid chromatography by the method of D. W. Kellog, J.Dairy Science, 52, 1690, 1969. Peak heights for acetic, propionic andbutyric acids are determined for samples from untreated and treatedincubation flasks.

When tested by this in vitro procedure, Antibiotic UK-58,852 at thelevel of 10 micrograms per milliliter gave rise to an increase of about93% in the production of propionic acid over that produced in thecontrol solution without added Antibiotic UK-58,852. By comparison thecommercially available compound salinomycin (another polycyclic etherantibiotic) at 10 mcg/ml. produced about an 88% increase of propionicacid over the control. In like manner, in a separate experiment,antibiotic CP-70,228 at a level of 10 mcg/ml. gave rise to an increaseof about 30% in propionic acid production, as compared to an increase ofabout 40% using salinomycin at 10 mcg/ml.

These data show that Antibiotics UK-58,852 and CP-70,228 will improvefeed utilisation by ruminants such as cattle and sheep. The compoundswill also have a similar effect in monogastric animals such as pigs andrabbits. Antibiotics UK-58,852 and CP-70,228 may be incorporated in feedcompositions as the free acid, sodium salt, potassium salt or mixturesthereof. Crude forms of Antibiotic UK-58,852 or CP-70,228, or driedfermentation broth containing the antibiotic, may also be incorporatedin feed compositions at the desired potency concentrations.

The in vitro antibacterial activity of the minor antibiotic componentsCP-70,228 and CP-70,828 is summarized in Table III:

                  TABLE III                                                       ______________________________________                                        ANTIBACTERIAL ACTIVITY                                                        MIC, mcg./ml.                                                                                Strain  CP-70,228  CP-70,828                                   Organism       No.     Sodium Salt                                                                              Sodium Salt                                 ______________________________________                                        Staphylococcus aureus                                                                        01A005  3.12       25                                                         01A052  3.12       25                                                         01A110  3.12       25                                                         01A400  6.25       25                                          Streptococcus faecalis                                                                       02A006  50         >50                                         Streptococcus pyogenes                                                                       020203  0.10       3.12                                        Actinomyces pyogenes                                                                         14D011  0.34                                                   Actinobacillus 54B004  >100                                                   pleuropneumoniae                                                              Pasteurella multocida                                                                        59A006  >100                                                   Clostridium perfringens                                                                      10A009  1.58                                                   Bacteroides fragilis                                                                         78C024  12.5                                                   Fusobacterium  84C004  >100                                                   necrophorum                                                                   Treponema hyodysenteriae                                                                     94A007  1.58                                                   ______________________________________                                    

Against the gram-negative bacteria such as Escherischia coli,Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens andEnterobacteriaceae aerogenes, MIC values were >50 in each case.

Efficacy data for Antibiotic Compound CP-70,228 against coccidialinfections in chickens following the procedures previously described issummarised in Table IV:

                  TABLE IV                                                        ______________________________________                                        IN VIVO ANTICOCCIDIAL ACTIVITY                                                CP-70,228                                                                                DOSE     AVERAGE                                                   INFECTION  (PPM IN  DEGREE OF   WEIGHT GAIN                                   SPECIES    FEED)    INFECTION   (PERCENT)                                     ______________________________________                                        Eimeria tenella                                                                          100      0.0         95                                                       75       0.3         63                                                       50       1.3         103                                                      25       2.7         80                                            Eimeria acervulina                                                                       100      0.0         100                                                      75       0.0         100                                                      50       0.0         70                                                       25       1.2         40                                            ______________________________________                                    

The invention is further illustrated by the following Examples.

EXAMPLE 1 1. Preparation of Inoculum

A sterile aqueous medium having the following composition was prepared.

    ______________________________________                                        Ingredient       Grams/liter                                                  ______________________________________                                        Glucose          20                                                           Soy Flour        10                                                           NZ Amine YTT*    5                                                            Sodium Sulphate  0.5                                                          Cobalt Chloride  0.002                                                        Calcium Carbonate                                                                              2                                                            ______________________________________                                    

One hundred ml of medium is distributed into 300 ml shake flasks andsterilized at 120° C. and 15 p.s.i. for 30 minutes. After cooling, themedium is inoculated with a vegetative cell suspension from a slantculture of Actinomadura sp. ATCC 39697 (N956-33).

The flasks are shaken at 28° C. on a rotary shaker having a displacementof 11/2 to 21/2 inches and 150 to 200 cycles per minute for three tofive days.

The following medium was also employed with similar results.

    ______________________________________                                        Ingredient       Grams/liter                                                  ______________________________________                                        Cerelose         10                                                           Corn Starch      5                                                            Corn Steep Liquor                                                                              5                                                            NZ Amine YTT*    5                                                            Cobalt Chloride  0.002                                                        Calcium Carbonate                                                                              3                                                            ______________________________________                                         *Registered trademark for enzymatic digest of casein, Humko Sheffield         Chemical Co. Inc.                                                        

2. Fermentation and isolation of UK-58,852

A shake flask containing the grown culture was used to inoculate a fiveliter fermentation vessel containing three liters of sterile medium ofthe following composition to which 1 ml of silicone antifoaming agenthad been added:

    ______________________________________                                        Ingredient         Grams/liter                                                ______________________________________                                        Cerelose           10.0                                                       Corn Starch        20.0                                                       NZ Amine YTT       5.0                                                        Wheat Germ         5.0                                                        Calcium Carbonate  4.0                                                        Water to 1 liter pH 6.9-7.0                                                   ______________________________________                                    

Fermentation was carried out at 30° C. with stirring at 1700 revolutionsper minute and aeration at one volume air per volume of broth per minuteuntil substantial activity was observed (based on antibiotic disc assayversus B. subtilis ATCC 6633), usually 4-6 days. The bioactivity of thebroth, and of subsequent recovery streams, were followed by using asensitive strain of Bacillus subtilis ATCC 6633 or Staphyloccocus aureusATCC 6538. The antibiotic component in the broth and recovery streamswas visualized by using silica gel plates developed with ethyl acetate.The plates were sprayed with vanillin reagent (3 g vanillin in 75 mlethanol and 25 ml 85% phosphoric acid) and heated at 80° C. AntibioticUK-58,852 appears as an orange to red spot. Alternatively, the plate wasoverlayed with agar, seeded with either S. aureus or B. subtilis, towhich 1.0 ml of a 1% solution of 2,3,5-triphenyl-2H-tetrazolium chloridehad been added, and incubated at 37° C. for 16 hours to visualize theantibiotic as a white area against a pink background.

At the end of this time the whole broth was extracted withmethylisobutyl ketone, the solvent separated and concentrated to yield35 g residue. The residue was suspended in hexane and batch treated withsilica gel on a filter funnel. The absorbant was washed with hexane,then eluted with chloroform, ethyl acetate and acetone. The activity wasin the ethyl acetate fraction (20 g). This was concentrated, the residuerechromatographed on silica and the product crystallized from heptane togive Antibiotic UK-58,852 as a white solid (yield 80 mgs).

EXAMPLE 2

The procedure of Example 1 was followed but using the following mediumfor the fermentation:

    ______________________________________                                        Ingredient       Grams/liter                                                  ______________________________________                                        Cerelose         10                                                           Corn Starch      5                                                            Corn Steep Liquor                                                                              5                                                            Cobalt Chloride  0.002                                                        NZ Amine YTT     5                                                            Calcium Carbonate                                                                              3                                                            ______________________________________                                    

The whole broth of a 10 pot fermentation of a culture of Actinomadurasp. ATCC 39697 (total volume approximately 25 liters) was extracted withone third volume of methylisobutyl ketone. The extract was concentratedunder vacuum to a brown oil (35 g). This material was chromatographed ona 5×100 cm column packed with column grade silica gel G (70-230 mesh,Woelm) in chloroformacetone (3:1). The column was developed withchloroform-acetone (3:1) at a flow rate of 10 ml/min. Fractions of 10 mleach were taken.

The fractions were examined by thin-layer chromatography on Analtechsilica gel GF plates developed in ethyl acetate. The plates were sprayedwith 3% vanillin in ethanol-85% phosphoric acid (3:1) and heated to 80°C. The desired antibiotic UK-58,852 appears as a red spot under theseconditions.

The fractions containing Antibiotic UK-58,852 were combined (totalvolume approximately 350 ml) and stirred with 2 grams of Darco G60carbon for 5 minutes. The mixture was filtered and evaporated undervacuum. The yellow oil remaining after evaporation was dissolved in 100ml of chloroform and stirred with an equal volume of water and the pHwas adjusted to 4.0 with phosphoric acid. The phases were separated andthe chloroform phase was stirred with an equal volume of 5% sodiumphosphate dibasic buffer and the pH was adjusted to 9.0 with 1N sodiumhydroxide solution. The phases were separated and the chloroform wasdried over anhydrous sodium sulphate and then evaporated under vacuum.The yellow viscous oil remaining after evaporation was dissolved in asmall volume of heptane whereupon crystallization occurred. The crystalswere collected by filtration and dried under vacuum yielding 3.5 g ofantibiotic UK-58,852 as the sodium salt; m.p. 182°-183° C. Found: C,60.90; H, 8.32; C₅₂ H₈₇ O₁₈ Na requires C, 61.06; H, 8.51. Ultravioletspectrum showed end absorption only. Optical rotation [α]_(D) =+10°(c=0.5, methanol). Infrared spectrum (KBr) cm⁻¹ : 3423, 2966, 2928,2869, 2821, 1613, 1453, 1407, 1377, 1361, 1312, 1257, 1238, 1206, 1175,1160, 1116, 1093, 1064, 1030, 983, 969, 938, 919, 886.

The free acid form of Antibiotic UK-58,852 was prepared by stirring achloroform solution of the sodium salt of UK-58,852 with an equal volumeof water and lowering the pH to 3.0 with phosphoric acid. The phaseswere then separated, and the chloroform was evaporated under vacuum togive Antibiotic UK-58,852 as the free acid: m.p. 123°-126° C.Ultraviolet spectrum showed end absorption only. Optical rotation[α]_(D) =+40° (c=0.5, chloroform).

EXAMPLE 3

A 1700 gallon fermentor, containing 1000 gallons of sterile medium ofthe following composition was inoculated with one liter of an inoculumof Actinomadura sp. ATCC 39697, prepared as described in Example 1.

    ______________________________________                                        Fermentation Medium                                                           Ingredient       Grams/liter                                                  ______________________________________                                        Cerelose         10                                                           Corn Starch      5                                                            Corn Steep Liquor                                                                              5                                                            Cobalt Chloride  0.002                                                        NZ Amine YTT     5                                                            Calcium Carbonate                                                                              3                                                            ______________________________________                                    

The fermentor was maintained at 28° C., with aeration and stirring at1700 revolutions per minute. After 296 hours the whole broth wasextracted with 300 gallons, followed by 200 gallons of methylisobutylketone. The solvent extracts were separated combined and concentratedunder vacuum to 10 liters of a brown oil. The extract was triturated bystirring with methanol for five minutes. The methanol phase wasseparated and the oil stirred with an additional 6 liters of methanol.The methanol fractions were combined and evaporated under vacuum (4liters). One liter of this methanol concentrate was taken up in 2 litersof hexane and poured onto a bed of 3 kg of column grade silica gel in aLapp filter. The silica gel was washed successively with hexane (11liters), chloroform (7.5 liters) and finally ethyl acetate (7.5 liters).The fractions were examined by thin layer chromatography. The productwas found to be in the chloroform and ethyl acetate fractions. Thechloroform and ethyl acetate fractions were both washed with watercontaining phosphoric acid to pH 4 and then with 5% sodium phosphatedibasic buffer (adjusted to pH 9.5 with 1N sodium hydroxide). Thesolvent fractions were then dried over anhydrous sodium sulphate andevaporated under vacuum. The oily residue remaining after evaporationwas taken up in a small amount of hexane, whereupon crystallizationoccurred. The crystals were collected by filtration. The remaining 3liters of methanol concentrate were treated as described above and thecrystals obtained from all four runs were combined, yielding 300 g ofAntibiotic UK-58,852 as the sodium salt.

EXAMPLE 4 Isolation of CP-70,228 from fermentation mother liquor

25 gm. of mother liquor from a UK-58,852 crystallization waschromatographed on a 50 mm×1 m column packed with column grade silicagel in a mixture of chloroform and acetone (4:1). The column was elutedat 10 ml/min with the same solvent, taking 10 ml fractions. Progress ofthe chromatography was monitored by thin layer chromatography. TheUK-58,852 was contained in fractions 95-230. After 300 fractions 25%methanol was added to the system and elution was continued for anadditional two liters. The CP-70,228 was contained in these fractions,which were evaporated to give 4.5 gm of a yellow oil. This material wasfurther chromatographed on a 25 mm×50 cm column of Sephadex LH-20 inmethanol. The flow rate was 1 ml/min and fractions were taken every 5minutes. Fractions shown by thin layer chromatography to containCP-70,228 were evaporated to give 1.3 g of a yellow oil. Thisconcentrate was re-chromatographed on a 25 mm×50 cm column packed withcolumn grade silica gel in chloroform containing 2.5% methanol. Thecolumn was run at 5 ml/min and fractions were taken every 2 minutes.Fractions shown by thin layer chromatography to contain CP-70,228 werecombined and evaporated to yield 100 mg of white solid CP-70,228. m.p.113°-123° C. Found: C, 62.63; H, 8.82. C₅₃ H₉₀ O₁₈ requires C, 62.70; H,8.94%. Ultraviolet spectrum: no absorption. Optical rotation: [α]_(D)=+21.8° (c=0.5, methanol). Infrared spectrum (KBr) cm⁻¹ : 3439, 2970,2933, 2877, 1716, 1636, 1457, 1380, 1318, 1293, 1240, 1218, 1165, 1117,1097, 1069, 1032, 989, 975, 940, 912, 897, 859.

EXAMPLE 5 Conversion of UK-58,852 to P-70,228 Sodium Salt

11 gm of UK-58,852 free acid was dissolved in 200 ml of methanol andallowed to stand at room temperature. Thin layer chromatography of thesolution after 1 hour showed complete conversion of the UK-58,852 toCP-70,228. The methanol was evaporated under vacuum and the concentratewas dissolved in 200 ml of chloroform. The chloroform, was stirred with200 ml of 5% sodium phosphate dibasic buffer and the pH was adjusted to10.5 with 6N sodium hydroxide. The phases were separated and thechloroform was dried over anhydrous sodium sulphate, filtered andevaporated under vacuum to an oil. 200 ml of heptane was added and thesolution was again evaporated under vacuum, whereupon the CP-70,228crystallized as the sodium salt. The salt was dried under high vacuum at60° C. for 5 hours, yield 11 g. Spectral data and chromatographyindicate that this material is identical to fermentation-derivedCP-70,228. m.p. 150°-155° C. Found: C, 60.49; H, 8.75. C₅₃ H₈₉ O₁₈ Narequires C, 61.37; H, 8.65%. Ultraviolet spectrum: no absorption.Optical rotation [α]_(D) =+13.6° (c=0.5, methanol). Infrared spectrum(KBr) cm⁻¹. 3429, 2973, 2935, 2878, 1594, 1455, 1399, 1379, 1319, 1291,1268, 1242, 1215, 1189, 1165, 1117, 1096, 1068, 1030, 990, 975, 943,891, 860, 833.

EXAMPLE 6 Isolation of CP-70,828 from fermentation mother liquor

The minor component CP-70,828 was isolated by repeated columnchromatography of fermentation mother liquors. CP-70,828 was detected byvanillin spray in the same manner as CP-70,228. Single-spot (by tlc)CP-70,828 material was crystallized from heptane, yield 20 mg. m.p.133°-136° C. Found: C, 62.66; H, 8.85. C₅₄ H₉₂ O₁₈ requires C, 63.01; H,9.01%. Ultraviolet spectrum: no absorption. Infrared spectrum (KBr) cm⁻¹: 3416, 3259 (br), 2968, 2926, 2871, 1614, 1455, 1377, 1363, 1315, 1260,1238, 1206, 1175, 1160, 1116, 1092, 1063, 1030, 986, 970, 939, 921, 887,857, 827, 799, 661.

We claim:
 1. An antibiotic of the formula: ##STR3## and pharmaceuticallyacceptable cationic salts thereof, wherein either R and R¹ are eachhydrogen; R is hydrogen and R¹ is methyl; or R and R¹ are both methyl.2. An antibiotic according to claim 1 in the form of its sodium orpotassium salt.
 3. The antibiotic according to claim 1, wherein R and R¹are each hydrogen.
 4. A nutrient feed composition for poultrycharacterized in that said feed comprises an antibiotic compoundaccording to claim 1 in an amount effective to control coccidialinfections in said poultry.
 5. A method for controlling coccidialinfections in poultry which comprises administering to said poultry ananticoccidially effective amount of an antibiotic compound according toclaim
 1. 6. A method according to claim 5 wherein said antibiotic isadministered to said poultry by adding said antibiotic to feed ingestedby the poultry.